AIDS vaccine candidates based on recombinant vectors may ultimately fail to provide adequate protection against HIV-1 in ongoing clinical trials. Live, attenuated vaccines afford more reliable protection in animals, but justifiable concerns about their safety will likely preclude their use in humans for the foreseeable future. Thus, the search continues for an AIDS vaccine that optimizes both safety and efficacy. One promising approach is the use of lentiviruses that are limited to a single cycle of infection. We have developed a unique system for producing strains of SIV that are limited to one round of infection as a non-replicating vaccine approach. Cells infected with this single-cycle SIV (scSIV) express all of the viral gene products except for Pol and release immature virus particles that cannot complete subsequent rounds of infection. In preliminary studies, rhesus macaques immunized with scSIV made antibody and T cell responses to multiple viral antigens. Following an intravenous challenge with SIVmac239, a strain that is notoriously difficult to protect against by vaccination, these animals also exhibited significant reductions in both acute and chronic phase viral loads relative to unvaccinated control animals. We now propose to more fully evaluate the extent of protection that may be achieved by this novel vaccine approach. In specific aim 1, we will test two different scSIV vaccine regimens to compare the effects of frequency versus dose of inoculation on the development of virus-specific immune responses and the ability to contain infection after a pathogenic SIV challenge. In specific aim 2, we will test the hypothesis that the induction of neutralizing antibodies by envelope-modified strains of scSIV can enhance protection. In specific aim 3, we will determine if the site of scSIV immunization influences the mucosal homing properties of virus-specific T cell responses and the ability to protect against a mucosal SIV challenge. Associations between immune responses elicited by scSIV and the outcome of challenge will be analyzed for each specific aim to reveal potential mechanisms of protection.